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WO2007010240A2 - Procede d'analyse a rendement eleve - Google Patents

Procede d'analyse a rendement eleve Download PDF

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Publication number
WO2007010240A2
WO2007010240A2 PCT/GB2006/002664 GB2006002664W WO2007010240A2 WO 2007010240 A2 WO2007010240 A2 WO 2007010240A2 GB 2006002664 W GB2006002664 W GB 2006002664W WO 2007010240 A2 WO2007010240 A2 WO 2007010240A2
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Prior art keywords
platelets
platelet
sample
activation
platelet activation
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PCT/GB2006/002664
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WO2007010240A3 (fr
Inventor
Hans Deckmyn
Isabelle Salles
Alexandre Fontayne
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EUROPEAN CARDIOVASCULAR GENETICS FOUNDATION
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EUROPEAN CARDIOVASCULAR GENETICS FOUNDATION
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Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/7056—Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
    • G01N2333/70564—Selectins, e.g. CD62
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/222—Platelet disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/224—Haemostasis or coagulation

Definitions

  • This invention relates to methods for assessing platelet activation. Such methods can be used to diagnose whether a subject has an increased risk of bleeding or thrombosis, to screen for compounds that are agonists or antagonists of platelet activation, or to screen for defective platelets.
  • platelet aggregometry Assessment of platelet activation is classically performed by platelet aggregometry. This technique was originally described by Born 22 . It closely mimics the physiological process in which a stimulus induces an activation cascade that finally results in GPIIb/llla mediated platelet aggregation.
  • platelet aggregometry suffers from several serious drawbacks. Although results are obtained in real time, it takes around five minutes for full aggregation to occur. In view of the relatively short active ex vivo fife- time of platelets, this precludes study of large numbers of agonists or antagonists at different concentrations unless a large number of aggregometers is used in parallel. The results obtained are not always reproducible, and interpretation of the results is sometimes not straightforward.
  • platelet activation Other parameters of platelet activation, such as secretion of ATP (detectable by luminescence) or appearance of platelet activation markers
  • FACS fluorescence- activated eel! sorting
  • a method for assessing platelet activation which comprises: stimulating platelets in a platelet sample to cause activation of the platelets; capturing activated platelets from the platelet sample in preference to resting platelets; and detecting the captured activated platelets.
  • the selectivity of capture of activated platelets to resting platelets is at least 2:1 activated platelets to resting platelets, preferably at least 3:1, or 10:1, more preferably at least 100:1,
  • activated platelets Preferential capture of activated platelets may be achieved in any suitable manner. However, it is preferred that activated platelets are captured using a monoclonal antibody (or a fragment or derivative thereof) that binds selectively to activated platelets.
  • the monoclonal antibody binds to an activated platelet marker, such as an activated integrin (for example, the activated form of GPHb/llla or GPIa/lla), or a granular protein (for example, CD62P or CD63).
  • an activated platelet marker such as an activated integrin (for example, the activated form of GPHb/llla or GPIa/lla), or a granular protein (for example, CD62P or CD63).
  • PAC-1 Monoclonal antibodies that bind selectively to activated platelets are known.
  • PAC-1 is a monoclonal antibody that is commercially available from BD Biosciences (San Diego, California: Cat. No. 340507). It recognises an epitope on the GPllb/IIIa complex of activated platelets at or near the fibrinogen binding site.
  • the GPIIb/Jlla complex (also known as ⁇ llb/?3) is a platelet-specific member of the integrin family of heterodimeric adhesive protein receptors found on a variety of cell types.
  • the GPIIb/llla complex is located on the surface membrane of resting platelets.
  • Platelet activation induces a calcium-dependent conformational change in GPIIb/llla that exposes a ligand binding site. Binding of fibrinogen to the activated form of the GPIlb/flla receptor is required for platelet aggregation. PAC-1 binds only to activated platelets and appears to be specific for this recognition site within GPIIb/llla.
  • AK-4 antibody is a purified mouse anti-human monoclonal antibody that is commercially available from BD Biosciences (San Diego, California: for example, Cat. No. 551345). It reacts with CD62P (also known as P- selectin), the 14OkDa membrane glycoprotein formerly known as platelet activation-dependent granule membrane protein (PADGEM), or GMP-140. CD62P is an integral part of the ⁇ -granule membrane of platelets and is rapidly translocated to the platelet plasma membrane upon activation.
  • CD62P also known as P- selectin
  • P- selectin the 14OkDa membrane glycoprotein formerly known as platelet activation-dependent granule membrane protein (PADGEM), or GMP-140.
  • CD62P is an integral part of the ⁇ -granule membrane of platelets and is rapidly translocated to the platelet plasma membrane upon activation.
  • the monoclonal antibody (or fragment or derivative) is immobilised to a solid phase, such as a well of a microtitre plate.
  • the captured activated platelets may be detected in any suitable way, but they are preferably detected using a monoclonal antibody (or a fragment or derivative thereof) that binds selectively to platelets, but which does not differentiate between resting and activated platelets.
  • Suitable monoclonal antibodies for this purpose are antibodies against GPIb ⁇ . Examples of commercially available monoclonal antibodies that bind specifically to platelets but which do not differentiate between resting and activated platelets are: SZ2 from lmmunotech (Marseille, France) or Beckman Coulter, AK2 from Cymbus Biotechnology (Hampshire, United Kingdom) or BioSource International, or HIP1 from Pharmingen (Becton Dickinson).
  • the monoclonal antibody (or fragment or derivative) used to detect the captured activated platelets may be labelled with a detectable label, or it may be a primary antibody detectable by a labelled binding agent (which may be a secondary antibody).
  • the amount of captured activated platelets is quantitatively determined to assess platelet activation.
  • a method of the invention may be a quantitative sandwich enzyme-linked immunosorbent assay (ELISA), radioimmunossay (RIA), immunoradiometric assay (IRMA), fluorescent immunoassay (FlA), chemiluminescent immunoassay (CLIA), or mirror resonance assay.
  • ELISA quantitative sandwich enzyme-linked immunosorbent assay
  • RIA radioimmunossay
  • IRMA immunoradiometric assay
  • FlA fluorescent immunoassay
  • CLIA chemiluminescent immunoassay
  • mirror resonance assay Such methods allow high sensitivity assessments of platelet activation.
  • Methods of the invention are particularly advantageous because they can be used to determine simultaneously the effect of different concentrations of a platelet agonist on platelet activation.
  • the platelet sample is split into a plurality of sample portions, and the platelets of each sample portion are stimulated with a different concentration of the same platelet agonist at the same time.
  • a dose-response curve of platelet activation to concentration of platelet agonist can be generated.
  • the effect of a plurality of different agonists on platelet activation can be determined (preferably at the same time).
  • the platelet sample may be stimulated with a plurality of different agonists (preferably at the same time), or the platelet sample may be split into a plurality of sample portions, and the platelets of each sample portion stimulated with a different agonist (preferably at the same time).
  • Methods of the invention may also be used to assess inhibition of platelet activation by contacting the platelets with an antagonist of platelet activation.
  • platelets of a platelet sample are stimulated to cause activation of the platelets, and the platelets are contacted with an antagonist of platelet activation. Activated platelets are then captured and detected.
  • the platelets may be contacted with the antagonist before, during, or after stimulation.
  • the platelet sample is split into a plurality of sample portions, and the platelets of each sample portion are contacted with the same concentration of the same agonist, and a different concentration of the same antagonist.
  • a dose response curve of platelet activation to concentration of antagonist may then be generated.
  • the effect of a plurality of different antagonists on platelet activation can be determined (preferably at the same time).
  • the platelet sample may be contacted with a plurality of different antagonists (preferably at the same time), or the platelet sample may be split into a plurality of sample portions, and the platelets of each sample portion contacted with a different antagonist (preferably at the same time).
  • Platelets may be stimulated in any suitable manner.
  • the platelets are contacted with a platelet agonist, for example ADP, TRAP, a TXA 2 analogue (such as U46619), collagen, or Collagen Related Peptide (CRP).
  • a platelet agonist for example ADP, TRAP, a TXA 2 analogue (such as U46619), collagen, or Collagen Related Peptide (CRP).
  • Any suitable antagonist of platelet activation may be used.
  • An example is S-nitroso-N-acetyl penicillamine (SNAP).
  • Methods of the invention can be used to diagnose defects in platelet activation, or in inhibition of platelet activation.
  • Methods of the invention may be used to determine whether a subject has, or is at risk of having, an abnormal blood condition associated with an increased risk of bleeding.
  • a method of the invention is carried out on a platelet sample obtained from the subject to determine whether activation of platelets in the sample is reduced compared to normal platelet activation.
  • Platelets in the sample may be stimulated with a platelet agonist. If platelet activation is reduced compared to normal platelet activation, the subject is diagnosed as having, or being at risk of having, an abnormal blood condition associated with an increased risk of bleeding.
  • platelets in the sample may be stimulated with a platelet agonist, and contacted with a platelet antagonist (either before, during, or after stimulation). If platelet activation is reduced compared to normal platelet activation this indicates that the response of the platelets in the sample to the antagonist is greater than normal, and the subject is diagnosed as having, or being at risk of having, an abnormal blood condition associated with an increased risk of bleeding.
  • EBs easy bruisers
  • Such individuals may be normal healthy individuals, but the EB phenotype may be of major clinical significance in situations where normal haemostasis is challenged.
  • EB identification is also expected to be of clinical use in certain categories of patients.
  • Identification of the EB phenotype by a method of the invention is expected to be of value for the following categories of individuals:
  • i) Patients with a low platelet count ( ⁇ 50 x 10 9 /L or even ⁇ 100 x 10 9 /L in exceptional circumstances, i.e. brain surgery or liver biopsy) because of, reduced generation by the bone marrow in cancer (i.e. by infiltration of the bone marrow with cancerous cells or iatrogenic), increased platelet destruction (i.e. in patients with antibody-mediated thrombocytopenia's), or increased consumption (i.e. in patients with disseminated intravascular coagulation) or Hypersplenism; ii) Patients undergoing major surgical procedures known for a high risk of significant blood loss (i.e.
  • cardio-thoracic surgery orthopaedic surgery, certainly the category of redo's; organ transplantation, especially liver, heart and lung and possibly kidney; surgery of trauma patients; brain surgery inclusive imaging; vessel surgery); iii) Patients undergoing biopsies of liver, kidney or brain or other vital organs; iv) Patients with inherited bleeding disorder such as haemophilia A or B, Von Will brands' disease and other more rare forms of inherited coagulopathies or with acquired coagulopathies (i.e. liver disease, viral hepatitis); v) Patients receiving blood thinning drugs (anti-coagulants, antiplatelet drugs, thrombolytic drugs) to reduce the risk of venous and/or arterial thrombosis.
  • blood thinning drugs anti-coagulants, antiplatelet drugs, thrombolytic drugs
  • Cerebral bleeds or bleeds in other vital organs can cause life-long disability or even death; vi) Healthy individuals who give blood as a donor by routine donation or blood derivatives by apheresis technology. One of the most frequent complaints of donors is serious bruising post-donation at the site of venepuncture.
  • additional drugs i.e. DDAVP, Tranexamic Acid
  • platelet levels required to cover invasive procedures could be tailored better to the patient by determining the platelet functional phenotype; iii) In patients with low platelets because of antibody-mediated destruction of platelets. Treatment of autoimmune mediated thrombocytopenia is based on a combination of the platelet count and clinical signs of bleeding. However, in patients with a very low platelet counts ( ⁇ 20 x 10 9 /L) treatment is given to reduce the risk of serious bleeding, even in the absence of bleeding. In these patients expensive therapies or therapies with major side effects are prescribed to reduce the relatively small risk of bleeding.
  • Stratifying of these therapies on basis of the platelet functional phenotype may be possible; iv) The prophylactic treatment regime in haemophilia patients may be better tailored to the patient depending on platelet functional phenotype. In addition life-style advice could be modified depending on the platelet functional phenotype; v) Patients on blood thinning drugs (these include anti-coagulants, anti-platelet drugs or thrombolytic drugs, or novel anti-platelet drugs still under development and based on inhibitors of collagen mediated platelet activation, such as drugs directed against platelet GPVI (such as antibodies 10B12 described in Patent WO 03/054020 A2) or against any of the proteins in the signalling pathway downstream of GPVI (for example src family kinases such as fyn).
  • Patients with an EB phenotype may require reduced dosing with these drugs.
  • There are no effective antidotes for several of these drugs i.e. Clopridogel, GPKb/llla antagonists, Tissue plasminogen activator, Streptokinase). Overdosing is an issue of serious concern and the therapeutic window of several of these drugs is relatively narrow. Dosing schedules may be adjusted on the functional platelet phenotype.
  • many novel 'blood thinning' drugs are being trialled or are under development. Whether these novel drugs will enter the market will depend on their clinical effectiveness and more importantly on the incidence and severity of side effects
  • the platelet functional phenotype may be one of these.
  • Platelets from donors with an EB phenotype may be less effective in preventing bleeding in the recipient when compared with platelets from 'normal' donors.
  • Donors with EB phenotypes may be barred from giving platelets.
  • Methods of the invention may be used to determine whether a subject has, or is at risk of having, an abnormal blood condition associated with an increased risk of thrombosis.
  • a method of the invention is carried out on a platelet sample obtained from the subject to determine whether activation of platelets in the sample is increased compared to norma! platelet activation.
  • Platelets in the sample may be stimulated with a platelet agonist. If platelet activation is increased compared to normal platelet activation, the subject is diagnosed as having, or being at risk of having, an abnormal blood condition associated with an increased risk of thrombosis.
  • platelets in the sample may be stimulated with a platelet agonist, and contacted with a platelet antagonist (either before, during, or after stimulation). If platelet activation is increased compared to normal platelet activation this indicates that the response of the platelets in the sample to the antagonist is less than normal, and the subject is diagnosed as having, or being at risk of having, an abnormal blood condition associated with an increased risk of thrombosis.
  • ECs easy clotters
  • Such individuals may be normal healthy individuals, and the EC phenotype may be of major clinical significance in situations where increased risk of clot formation may cause severe disease, i.e. myocardial infarction (Ml), stroke or other thrombotic events in arterial circulation.
  • Ml myocardial infarction
  • EC identification is also expected to be of clinical use in certain categories of healthy individuals and in patients.
  • Identification of the EC phenotype by a method of the invention is expected to be of value for the following categories of individuals:
  • AT blood clots or thrombi in arteries
  • vessel occlusion which may be partial or complete
  • AT leads to suboptimal (or a lack of) perfusion of target tissue (the tissue to which the supplying blood vessel is blocked by AT) with blood. This leads to insufficient (or loss of) oxygen supply causing loss of function or irreversible damage with death of tissue.
  • target tissue the tissue to which the supplying blood vessel is blocked by AT
  • oxygen supply causing loss of function or irreversible damage with death of tissue.
  • Infarction (Ml), unstable and stable Acute Coronary Syndrome(ACS)); ii) Patients with AT in blood vessels of the brain with cerebral stroke; iii) Patients with AT in peripheral blood vessels (peripheral artery disease or PAD), i.e. of the legs resulting in intermittent claudication and/or chronic leg ulcers; iv) Patients with AT in blood vessels supplying other vital organs
  • Preventative measures In healthy individuals with a family history of Ml or of another disease associated with AT (i.e. stroke, PAD) use of simple preventative medication can be applied from an early age (i.e. daily use of aspirin) to substantially reduce the risk of disease. Moreover, life-style advice can be used to further modify disease risk (i.e. cessation of smoking, reducing body weight, increased exercise, change of diet). Defining the EC phenotype of platelets by a method of the invention may be of significant value to better tailor preventative measures to those groups in the population most at risk of AT. This is important from a public health perspective since reducing the chance of AT in individuals considered to be at increased risk (i.e.
  • An EC phenotype as defined by a method of the invention may lead to the lowering of the age limit for the use of preventative measures (i.e. aspirin, life-style changes) in case of a positive family history of AT related diseases and an EC phenotype.
  • preventative measures i.e. aspirin, life-style changes
  • Methods of the invention are expected to have widespread use since many individuals in the population are at risk of AT.
  • AT-related diseases are the main cause of death in Western Society and the cost of prevention is substantial. With improved diagnosis of individuals at risk, overall health care system costs can be reduced; Acute measures. If a patient is diagnosed with an acute AT- related disorder (such as Ml or stroke) treatment needs to be instigated with urgency.
  • Treatment in the acute phase consists of combinations of thrombolytic drugs, anti-platelet drugs and other drugs (i.e. lipid lowering drugs, beta-blockers, angiotensin converting enzyme inhibitors).
  • Cocktails of anti-platelet drugs i.e. aspirin, Clopridogel and GPIIb/llla inhibitors
  • bleeding is one of the major side effects of combination anti-platelet drug therapy in up to 2% of patients.
  • the application of multiple anti-platelet drugs may be warranted but for patients with EB phenotypes use of multiple anti-platelet drugs may be contraindicated.
  • Methods of the invention may aid the stratification of drug therapy in the acute phase of AT-related disorders reducing the risk of post-event complications
  • Blockade of platelet activation with anti-platelet drugs is considered to be of benefit in the pre- intervention and peri-interventfona! treatment by angioplasty. I.e. there is growing recognition that such treatment is useful for NSTEMi (Non ST-Elevated Ml, i.e. patient with a heart attack without effects on the electrocardiogram that have been regarded as diagnostic, still with elevated creatine kinase, etc.). These patients are at high risk, and will be scheduled for angioplasty, and are put on anti-platelet drugs whilst they are waiting. However, as mentioned anti-platelet drugs are not without risks and stratification of use may be informed by the platelet phenotype as diagnosed by a method of the invention.
  • Normal platelet activation may be taken as a known value for normal platelets, or by performing the method on the platelet sample in parallel with a sample of normal platelets.
  • Methods of the invention may also be used in screening assays to identify compounds (for example from compound libraries) that are agonists or antagonists of platelet activation, or that enhance platelet activation. Such compounds may have therapeutic use for the treatment of abnormal blood conditions associated with an increased risk of bfeeding or thrombosis.
  • a screening assay to screen for an antagonist of platelet activation which comprises: stimulating platelets in a platelet sample; contacting the platelets with a candidate antagonist; capturing activated platelets from the platelet sample in preference to resting platelets; detecting the captured activated platelets to assess platelet activation; and identifying and/or isolating the candidate antagonist if platelet activation is antagonised by the candidate antagonist.
  • the platelets should be stimulated with a known platelet agonist (for example, any of the known agonists listed above).
  • the candidate antagonist may be contacted with the platelets before, during, or after stimulation of the platelets, but preferably before or during stimulation.
  • a screening assay to screen for an agonist of platelet activation which comprises: contacting platelets in a platelet sample with a candidate agonist; capturing activated platelets from the platelet sample in preference to resting platelets; detecting the captured activated platelets to assess platelet activation; and identifying and/or isolating the candidate agonist if platelet activation is agonised by the candidate agonist.
  • a screening assay to screen for a compound that enhances platelet activation comprises: stimulating platelets in a platelet sample to cause activation of the platelets; contacting the platelets with a candidate enhancer of platelet activation; capturing activated platelets from the platelet sample in preference to resting platelets; detecting the captured activated platelets to assess platelet activation; and identifying and/or isolating the candidate enhancer if platelet activation is enhanced by the candidate enhancer.
  • the candidate enhancer may be contacted with the platelets before, during, or after stimulation of the platelets, but preferably before or during stimulation.
  • Methods of the invention may also be used to screen for defective platelets (either hypo- or hyper-reactive platelets).
  • a screening assay to screen for defective platelets which comprises: stimulating platelets in a plurality of different platelet' samples; capturing activated platelets from the platelet samples in preference to resting platelets; detecting the captured activated platelets to assess platelet activation; and identifying the platelet sample as containing defective platelets if platelet activation is reduced or enhanced compared to normal platelet activation.
  • Defective platelets identified by a screening assay of the invention may be further investigated to identify risk genes for cardiovascular diseases, in particular genes associated with an increased risk of bleeding or thrombosis.
  • Methods of the invention may readily be used in a high-throughput format to determine simultaneously the effect of several different concentrations of agonists or antagonists on platelet activation, or of several different agonists or antagonists on platelet activation, or to screen simultaneously several different candidate compounds to identify agonists, antagonists, or enhancers of platelet activation.
  • methods of the invention are carried out using a microtitre plate in which a monoclonal antibody used to capture activated platelets is immobilised to each well of the microtitre plate.
  • a monoclonal antibody used to capture activated platelets is immobilised to each well of the microtitre plate.
  • kit for assessing platelet activation which comprises: a platelet agonist for stimulating platelets in a platelet sample to cause activation of the platelets; a capturing agent for capturing activated platelets from the platelet sample in preference to resting platelets; and a detecting agent for detecting captured activated platelets.
  • kits of the invention may further comprise all reagents necessary to determine quantitatively the amount of captured activated platelets, for example labels or reagents required for controls or comparisons.
  • a kit of the invention may further comprise a platelet antagonist.
  • a kit of the invention may further comprise a solid phase to which the capturing agent is immobilised.
  • the solid phase comprises a plurality of sample wells or chambers, each sample well or chamber for receiving a portion of the platelet sample, and the capturing agent being immobilised to a wall of each sample well or chamber.
  • each sample well/chamber may comprise a different concentration of the same platelet agonist
  • each sample well/chamber may comprise a different concentration of the same platelet antagonist (optionally additionally with a platelet agonist: preferably the same concentration of platelet agonist in each sample well).
  • each sample well/chamber may comprise a different agonist or a different antagonist.
  • each sample well/chamber may comprise a different candidate agonist or enhancer of platelet activation, or a different candidate antagonist of platelet activation.
  • the different candidate agonists, enhancers, or antagonists may be different compounds of a compound library which is to be screened for the presence of agonists, enhancers, or antagonists of platelet activation.
  • kits comprising a solid phase with a plurality of sample wells/chambers may be used for high-throughput screening assays in accordance with the invention.
  • FIG 1 shows ADP-induced binding of platelets to 16N7C2 monoclonal antibody (moAb) (anti-GPllb/llla).
  • moAb monoclonal antibody
  • A-C 5 ⁇ g/ml 16N7C2 was coated in a 96-well microtitre plate overnight at 4°C and subsequently incubated with platelets (PRP) in the presence of 0-20 ⁇ M ADP. Platelet binding was detected by addition of biotinylated-6B4 moAb (anti-GP!b ⁇ )/streptadivin- HRP.
  • Sigmoidal regression fit was applied to the curves using ORIGIN software. Each curve represents data from one donor prepared in duplicate (Mean ⁇ SE).
  • FIG 2 shows TRAP (thrombin receptor agonist peptide)-induced binding of platelets to 16N7C2 moAb (anti-GPllb/llla).
  • A-C 5 ⁇ g/ml 16N7C2 antibody was coated in a 96-weiI microtitre plate overnight at 4°C and subsequently incubated with platelets (PRP) in the presence of 0-40 ⁇ M TRAP. Platelet binding was detected by addition of biotinylated-6B4 moAb (anti-GP!b ⁇ )/streptadivin-HRP.
  • Sigmoidal regression fit was applied to the curves using ORIGIN software. Each curve represents data from one donor prepared in duplicate (Mean ⁇ SE);
  • FIG. 3 shows U46619- and collagen-induced binding of platelets to
  • 16N7C2 moAb (anti-GPllb/llla). 5 ⁇ g/ml 16N7C2 antibody was coated in a
  • FIG. 4 shows ADP-induced binding of platelets to SZ51 moAb (anti-P- sefectin).
  • A 5 ⁇ g/ml SZ51 antibody was coated in a 96-well microtitre plate overnight at 4°C and subsequently incubated with platelets (PRP) in the presence of 0-20 ⁇ M ADP. Platelet binding was detected by addition of biotinylated-6B4 moAb (anti-GPIb ⁇ )/streptadivin. Sigmoidal regression fit was applied to the curves using ORIGIN software. Each curve represents data from one donor prepared in duplicate (Mean ⁇ SE).
  • B Binding of SZ51 antibody to resting platelets (grey area) and platelets stimulated with 20 ⁇ M ADP (black line) analyzed by flow cytometry. For control, cf. Figure 1D;
  • Figure 5 shows inhibition of binding of platelets to 16N7C2 moAb (anti- GPIlb/Illa).
  • A 5 ⁇ g/ml 16N7C2 antibody was coated in a 96-well microtitre plate overnight at 4°C and subsequently incubated with platelets (PRP) in the presence of 0-20 ⁇ M ADP. PRP from the same donor was isolated before (closed circles) and 1 hour after aspirin intake (open triangles). Platelet binding was detected by addition of biotinylated-6B4 moAb (anti- GPIb ⁇ )/streptadivin.
  • B PRP was incubated with 10 ⁇ M ADP with variable amount of antagonist SNAP 0-2 mM. Platelet binding was revealed as described above.
  • CAD coronary artery disease
  • moAbs monoclonal antibodies
  • Platelet-rich plasma was prepared from blood from healthy donors using citrate as anticoagulant, and platelets were stimulated with different agonists, Including ADP, TRAP (thrombin receptor agonist peptide), U46619 (thromboxane A2 analogue) or collagen. Levels of platelet activation were assessed using a quantitative sandwich ELISA where moAbs 16N7C2 or SZ51 raised against platelet receptors GPIIb/llla and P- selectin, respectively, were used to capture activated platelets. The biotinylated mouse anti-GPIb ⁇ moAb 6B4 was used to reveal the presence of platelets.
  • GPIIb/llla and P-selectin dependent signals were significantly 1.5-3-times higher when platelets were stimulated as compared with resting platelets.
  • Prior incubation of platelets with the nitric oxide donor SNAP inhibited the capture via GPIIb/llla or P-selectin induced by sub- maximal concentrations of agonists.
  • platelets taken one hour after aspirin intake could no (onger be activated.
  • Intracellular calcium mobilization triggers extracellular influx which amplify GPIlb/IIIa activation, resulting in firm platelet adhesion 9 .
  • platelets firmly adhere to collagen via the main collagen receptors GPVl and integrin ⁇ 2 ⁇ i, triggering a critical signalling that results in platelet activation.
  • the GPVI receptor acts through tyrosine kinases (Fyn and Lyn) that phosphorylate ITAM domains on FcR ⁇ -chain, which in turn activate Syk, triggering an activation cascade involving adaptor proteins LAT and SLP- 76.
  • PLC protein kinase C
  • P13- kinase phosphoinositol 3-kinase
  • PLC protein kinase C
  • TXA 2 is formed upon activation of phospho ⁇ pase A2 (PLA 2 ) 10 .
  • thrombin is one of the most potent activators of platelets via its receptors: protease-activated receptors (PAR) and GPIbMlX 11"14 .
  • PAR protease-activated receptors
  • GPIbMlX 11"14 GPIbMlX 11"14 .
  • Secreted agonists mentioned above except ATP and GAS6 cause platelet activation through G-protein coupled receptors (GPCRs).
  • the GPCR Gq family (PAR1 and PAR4 for thrombin, TPa for TXA 2 and P2Yi for ADP) activates PLC ⁇
  • the GPCR Gi family (PAR 1, PAR4, P2Yi 2 for ADP) inhibits adenyl cyclase and/or activates MAP-kinases
  • the GPCR G12/G13 family (PAR1 , PAR4, TPa) signals to small GTPases 10 .
  • the end point of platelet activation is aggregation in which integrin GPIIb/llla plays an important role by binding fibrinogen upon activation 15 .
  • MoAbs anti-GPlb ⁇ 6B4 and anti-GPIIb/llla 16N7C2 used in this study were produced and characterized as previously described 16 ' 17 .
  • the moAb SZ51 binds CD62P on activated platelets and was a generous gift from Dr.
  • MoAb 6B4 was biotinylated using NHS LC Biotin
  • MOPC-21 was purchased from Sigma (St Louis, MO). Detection of unlabelled antibodies MOPC21, 16N7C2 and SZ51 in flow cytometry was by a goat anti-mouse
  • PE IgG-phycoerythrin
  • 96-weil microtitre plates (Greiner, Frickenhausen, Germany) were coated overnight at 4° C with 100 ⁇ l/well moAb 16N7C2, or SZ51 (5 ⁇ g/ml in PBS). Plates were blocked with 3 % milk powder (250 ⁇ l/well) for 2 h at RT.
  • Agonists ADP (Sigma), TRAP (Calbiochem), TXA 2 analogue U46619 (Caibiochem) and Horm collagen (Nycomed, Kunststoff, Germany) were prepared in 25 ⁇ l by a serial dilution in PBS with starting concentrations of 20 ⁇ M, 40 ⁇ M, 30 ⁇ M and 150 ⁇ g/ml, respectively.
  • the antagonist S- nitroso-N-acetyl penicillamine (SNAP; Calbiochem) at 2 mM was prepared in 25 ⁇ l by a serial, dilution in PBS with a constant concentration of ADP (10 ⁇ M). Immediately after the agonists/antagonists dilutions, 75 ⁇ l of PRP was added to each well and incubated at room temperature for 30 minutes. Every condition was tested in duplicate. Biotinylated moAb 6B4 was used as a primary antibody for platelet detection at 1 ⁇ g/ml in PBS- 0,3% milk powder for 1 hour at room temperature.
  • MoAb 6B4 binding to captured platelets was revealed with streptavidin coupled with horse radish peroxidase (Roche, Mannheim, Germany) (1/5000 in PBS, 0.3% milk, 1 h at RT). The colour reactions were initiated by adding H 2 O 2 and orthophenylenediamine (OPD; Sigma) and stopped with 4M H 2 SO 4 . Platelet binding was evaluated by measuring the absorbance at 490 nm.on a m ⁇ crotitre plate reader (EL340, Bio-Tek instruments). After each incubation, a washing step was performed three times with PBS.
  • FACS analysis Platelets (5 x 10 6 ) were diluted in 200 ⁇ l PBS and incubated with 10 ⁇ M ADP, for 10 minutes at room temperature. At the end of the incubation, 1/5 of the reaction mixtures were first incubated for 15 minutes at RT with moAbs 16N7C2, SZ51 or MOPC-21. A secondary antibody PE-conjugated goat anti-mouse IgG was then added for 15 min before fixation with 0,2 % formyl saline (0,9 % NaCI, 0,2% formaldehyde). Samples were then read on a FACScan flow cytometer (EPICS ® XL-MCL, Coulter). A gate analysis on 10,000 events was performed based on forward and side scatter to exclude platelet aggregates and microparticles.
  • the moAb16N7C2 was the first anti-GPIIb/llla that contains an echistatin-like RGD sequence in one of its complimentarity determining regions (CDRs) (GDR3 of the heavy chain variable domain) ⁇ and with a high affinity for its antigen (GPllb/llia).
  • CDRs complimentarity determining regions
  • GPllb/llia high affinity for its antigen
  • the moAb SZ51 18 has been raised against P-selectin (CD62P) present in the alpha granules of resting platelets 19 .
  • CD62P P-selectin
  • the moAb 6B4 targets the region amino acids 230-240 on the alpha subunit of the , heterodimeric GPIb ⁇ platelet receptor. It is a well described inhibitor of the GPIb ⁇ -VWF interaction 16 . MoAb 6B4 was chosen to reveal the binding of platelets as it does not differentiate between resting and activated states, and furthermore GPIb ⁇ is present in fairly high copy number on the platelet surface (25,000 copies) 21 . Activation of platelets with various agonists led to an increase in platelet binding to the anti-GPilb/IIIa moAb as illustrated in Figures 1-3.
  • the ultimate design of the test could consist in one ELISA plate per donor/patient, for which around 20 ml blood would be required, which then would be sufficient to study 8 concentrations of 4 agonists and of 1 antagonist, plus controls, in duplicate.
  • the platelets are studied simultaneously such that variability due to different handling times is avoided, the data are produced in a digital form, making interpretation more straightforward, as the plates containing coated capturing antibody and the different concentrations of (ant)agonists can be prepared on beforehand and stored frozen, the remainder of the test can readily be automated, and finally, and potentially the more important advantage could well be that the possibility to detect hyper-active platelets, i.e. reacting at lower than on average required agonist concentrations, is built-in.
  • a platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol.

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Abstract

Procédés d'évaluation d'activation de plaquettes, utilisables pour le diagnostic de risque accru d'hémorragie ou de thrombose, l'analyse de composés agonistes ou antagonistes vis-à-vis de l'activation de plaquettes. Procédé d'évaluation de cette activation ou d'analyse de plaquettes défectueuses : stimulation de plaquettes dans un échantillon de plaquettes pour en induire l'activation ; capture des plaquettes activées à partir de l'échantillon de préférence par rapport aux plaquettes au repos ; et détection des plaquettes activées capturées.
PCT/GB2006/002664 2005-07-18 2006-07-18 Procede d'analyse a rendement eleve Ceased WO2007010240A2 (fr)

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US7790410B2 (en) 2008-04-25 2010-09-07 General Electric Company Method and apparatus for determining hemocompatibility
CN103645320A (zh) * 2013-11-19 2014-03-19 洛阳莱普生信息科技有限公司 一种呋喃它酮代谢物化学发光酶联免疫快速检测试剂盒
CN103645322A (zh) * 2013-12-12 2014-03-19 洛阳莱普生信息科技有限公司 一种链霉素的化学发光酶联免疫检测试剂盒
CN103675273A (zh) * 2013-11-12 2014-03-26 洛阳莱普生信息科技有限公司 一种犬瘟热病毒抗原化学发光检测试剂盒
CN103675286A (zh) * 2013-12-12 2014-03-26 洛阳莱普生信息科技有限公司 一种林可霉素化学发光酶联免疫快速检测试剂盒
CN103675287A (zh) * 2013-12-12 2014-03-26 洛阳莱普生信息科技有限公司 一种伊维菌素化学发光酶联免疫快速检测试剂盒
CN103728450A (zh) * 2013-11-08 2014-04-16 洛阳莱普生信息科技有限公司 一种检测盐酸克伦特罗化学发光检测试剂盒
WO2021224236A1 (fr) * 2020-05-05 2021-11-11 Etablissement Français Du Sang Methode de detection de l'activation plaquettaire liee a l'inflammation
CN115737814A (zh) * 2022-10-26 2023-03-07 中国科学院大学 抗血小板药物及制备方法、抗血小板药物捕获剂

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WO1994013307A1 (fr) * 1992-12-11 1994-06-23 Steritech, Inc. Activation et fonction plaquettaires
WO2000043793A1 (fr) * 1999-01-26 2000-07-27 Du Pont Pharmaceuticals Company PROCEDE A FACS PERMETTANT LA DETECTION D'ACTIVATEURS DEPENDANTS DE L'INHIBITEUR GPIIb/IIIa DANS DES ECHANTILLONS DE PLASMA

Cited By (10)

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Publication number Priority date Publication date Assignee Title
US7790410B2 (en) 2008-04-25 2010-09-07 General Electric Company Method and apparatus for determining hemocompatibility
CN103728450A (zh) * 2013-11-08 2014-04-16 洛阳莱普生信息科技有限公司 一种检测盐酸克伦特罗化学发光检测试剂盒
CN103675273A (zh) * 2013-11-12 2014-03-26 洛阳莱普生信息科技有限公司 一种犬瘟热病毒抗原化学发光检测试剂盒
CN103645320A (zh) * 2013-11-19 2014-03-19 洛阳莱普生信息科技有限公司 一种呋喃它酮代谢物化学发光酶联免疫快速检测试剂盒
CN103645322A (zh) * 2013-12-12 2014-03-19 洛阳莱普生信息科技有限公司 一种链霉素的化学发光酶联免疫检测试剂盒
CN103675286A (zh) * 2013-12-12 2014-03-26 洛阳莱普生信息科技有限公司 一种林可霉素化学发光酶联免疫快速检测试剂盒
CN103675287A (zh) * 2013-12-12 2014-03-26 洛阳莱普生信息科技有限公司 一种伊维菌素化学发光酶联免疫快速检测试剂盒
WO2021224236A1 (fr) * 2020-05-05 2021-11-11 Etablissement Français Du Sang Methode de detection de l'activation plaquettaire liee a l'inflammation
FR3109999A1 (fr) * 2020-05-05 2021-11-12 Etablissement Français Du Sang Methode de detection de l’activation plaquettaire liee a l’inflammation
CN115737814A (zh) * 2022-10-26 2023-03-07 中国科学院大学 抗血小板药物及制备方法、抗血小板药物捕获剂

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